The goal of our studies is to understand the mechanisms that regulate viral late gene expression and genome amplification during the productive life cycle of oncogenic human papillomaviruses (HPVs). Papillomaviruses are small DNA viruses that induce a variety of proliferative epithelial lesions. Infection by papillomaviruses occurs into the basal cells of the epithelia where genomes are established as low copy episomes. The life cycle of papillomaviruses is tightly linked to epithelial differentiation with the induction of late gene expression, genome amplification and assembly of virions restricted to suprabasal cells. My laboratory first developed methods to grow human papillomaviruses in tissue culture and extended these techniques to synthesize HPVs from cloned DNA. These methods have allowed for a detailed genetic analysis of the productive life cycle of human papillomaviruses. In the recent grant period we have identified sequences within the URR and the E6/E7 coding sequences that regulate late gene expression in differentiated cells. Our studies have also identified E2F2, C/EBP2, C/EBP1 as prime candidates to be the major regulators of late gene expression. Additional work demonstrated that HPV proteins activate caspases upon differentiation and that this is necessary for efficient genome amplification. We further determined that caspases target the E1 proteins and that this facilitates amplification. In this grant, I propose to investigate how late gene expression and amplification are regulated by asking the following questions: 1) How is HPV late gene expression regulated in differentiating epithelia? How do E2F2, C/EBP2, C/EBP1 function in the activation of late expression? Do viral proteins play a role in activating late expression independent of their role in mediating amplification? 2) What mechanisms regulate differentiation-dependent amplification? How do the caspase cleaved forms of E1 and E2 contribute to genome amplification in differentiating cells?